Method for testing the degradation of polymeric materials

ABSTRACT

The present invention provides a novel method for monitoring the surface concentration of a drug in a polymer blend matrix and the reaction kinetics of the biodegradable polymers. Detailed information on surface concentration, degradation rates, degradation kinetics and mechanism, is provided by using Time-of-Flight Secondary Ion Mass Spectrometry (ToF SIMS) measurements. Also provided is a method for determining oligomers in hydrolyzed biodegradable polymers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/680,701 filed Oct. 6, 2000 abandoned, which claims priorityto U.S. Provisional Patent Application No. 60/157,964, filed on Oct. 6,1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grants CHE9704996and CHE0079114 from the National Science Foundation. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of degradablepolymers. More particularly, the present invention provides a method fordetermining oligomeric degradation products of biodegradable polymerseither alone or simultaneously with surface concentration of drug inpolymer-drug blend matrices and the drug release characteristics of suchblends. This information is useful in determining degradation reactionkinetics of biodegradable polymers and biodegradable polymer-drug blendmatrices and the drug release characteristics of such blends. Syntheticbiodegradable polymers have been used in clinical applications fordecades. Some relevant applications include surgical implants, woundhealing materials, absorbable sutures and drug delivery devices. Amongissues important in developing biomedical applications based on polymerbiodegradability are the properties of degradation (such as rate,mechanism, by products, etc.) of the polymer material. The study ofhydrolytic degradation of biodegradable polymers has been a researchfocus in the past few decades with in vivo investigations of biopolymerimplants being the major clinical investigation method. Directmonitoring of the weight loss of polymer implants and histologicalobservations provides macroscopic information on the hydrolyticdegradation. A drawback of this method is that it is very timeconsuming.

For the in vitro investigation of hydrolytic degradation ofbiodegradable polymers, many bulk characterizations have been developed.Properties such as tensile strength, thermal properties, mass loss, anddecrease in molecular weight have been measured. Techniques usedinclude: differential scanning calorimetry (DSC), gravimetry, gelpermeation chromatography (GPC), size exclusion chromatography (SEC),FTIR, NMR, X-ray diffraction and laser diffractometry.

Among the surface sensitive microscopic and spectroscopic techniques,methods such as scanning electron spectroscopy (SEM) and atomic/scanningforce microscopy have become important means for studying biodegradationof polymers. The surface microscopic techniques, however, do not providechemical compositional or structural information.

A class of biodegradable polymer that has attracted considerableattention for the design of novel drug delivery systems is thepolyesters. These include poly-(α-hydroxy acids), poly(β-hydroxy acids),poly(α-malic acids), pseudopoly(α-amino acids), their copolymers, andmixtures thereof. Of particular interest are the polyesters with pendantcarboxylic acid groups. These carboxylic acid groups may befunctionalized to manipulate material properties and are thought to havea catalytic effect on the hydrolytic scission of the ester bonds,increasing the degradation rate.

The drug release kinetics from drug/biodegradable polymer blend matricesis complicated due to both polymer erosion and drug diffusion throughpreformed microporous channels within the matrices. Factors such asmorphology and crystallinity of a polymer, formulation, drug molecularsize and solubility may have significant influence not only on thedegradation of drug delivery devices, but also on the release profile ofa drug. Furthermore, it has been reported that it is difficult topredictably control drug release over a desired period. This issuspected as being due more to an initial burst (rapid release) of drugcombined with the process of relatively faster drug diffusion thanpolymer degradation of the matrices. Although a number of studies havebeen directed toward drug release profiles and correlating these resultswith polymer degradation kinetics, little attempt has been made tosimultaneously determine both, especially with respect to thesurface/interface chemistry for the induction phase of bulk erosion ofdrug/biodegradable polymer blend matrices.

Thus, conventional techniques do not provide information toquantitatively describe the initial burst of drug release with thepolymer degradation kinetics at the induction phase of bulk erosion ofthe blend matrices. Thus, there is a pressing need to develop powerfuland fast methods for evaluating and screening the degradation kineticsof biodegradable polymers and drug release profile in the inductionperiod of bulk erosion of biodegradable polymer blend matrices.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a novel method for simultaneouslydetermining both surface concentration of a drug and degradationkinetics of a biodegradable polymer/drug blend matrix by usingTime-of-Flight Secondary Ion Mass Spectrometry (ToF SIMS) measurements.The method comprises simultaneously determining surface concentration ofa drug in the polymer/drug matrix and reaction kinetics of a degradablepolymer in the polymer/drug matrix by the following steps: providing apolymer/drug matrix, initiating degradation of the polymer/drug matrix,subjecting the degraded polymer/drug matrix to high mass and low massToF SIMS spectral analysis, identifying and quantifying oligomers fromthe high mass ToF SIMS over a period of time, identifying andquantifying surface drug from the ToF SIMS spectra over the same periodof time, and calculating the rate of formation of one or more oligomersand the rate of change of surface concentration of drug.

In yet a further embodiment of the present invention, oligomers of ahydrolyzed biodegradable polymer are identified and quantified by thefollowing steps: providing a biodegradable polymer, initiatinghydrolytic degradation of the biodegradable polymer, subjecting thedegraded biodegradable polymer to high mass ToF SIMS spectral analysisand identifying and quantifying oligomers from the high mass ToF SIMSspectral analysis.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a representation of the three major steps in the course ofpolymer erosion.

FIG. 2 is a plot showing degradation kinetics at varying pH.

FIG. 3 is a representation of a high mass portion of the ToF SIMSspectra of hydrolyzed PGA.

FIG. 4 is a representation of a high mass portion of the ToF SIMSspectra of hydrolyzed PLA.

FIG. 5 is a representation of a high mass portion of the ToF SIMSspectra of hydrolyzed PLGA 50:50 copolymer.

FIG. 6 is a representation of the ToF SIMS spectra of hydrolyzed PLGAfrom 800 D to 1000 D. Number marked peaks are molecular ion peaks.

FIG. 7 is a comparison of a group of molecular peaks in the ToF SIMSspectra with the theoretically calculated mass spectra.

FIG. 8 is a representation of a high mass portion of the ToF SIMSspectra of hydrolyzed PSA.

FIG. 9 is a representation of a high mass portion of the ToF SIMSspectra of PFS 20:80 copolymer before hydrolysis treatment.

FIG. 10 is a representation of a high mass portion of the ToF SIMSspectra of hydrolyzed PFS 20:80 copolymer.

FIG. 11 is a representation of a high mass portion of the ToF SIMSspectra of hydrolyzed PES 50:50 copolymer.

FIG. 12 is a comparison of the low mass portion (90 D to 103 D) of theToF SIMS spectra of PFS copolymer before and after hydrolysis.

FIG. 13 is a plot of log molecular ion peak intensity of hydrolyzedpolyanhydrides versus degree of polymerization of the hydrolysisproducts.

FIG. 14 is a graphical representation of the concentration of Ph₃Naccumulated at the surface of Ph₃N/PLLA (20:80 wt%) blend matrices as afunction of hydrolysis time in two pH buffered conditions, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The expression “polymer blend matrix” and words of similar effect asutilized herein refer to a mixture of at least one polymeric componentand at least one non-polymeric component. Typical blend matrices willcontain between about 1 to about 99 and about 99 to about 1 wt% polymerto non-polymer.

The term “drug” as utilized herein to describe suitable blend matricesrefers to chemical compositions and is intended to be interpreted in itsbroadest sense. Therefore “drug” would include substances used in thediagnosis, treatment or prevention of disease or as a component ofmedication as well as other chemicals or dyes.

The term “degradation” as utilized herein refers to the disruption ofthe chemical linkages that form a polymeric blend matrix from a numberof one or more different monomeric subunits. Therefore degradation maybe a result of chemical activities such as salvation, desorbtion,dissociation, hydrolysis, dissolution, oxidation, reduction, photolysis,etc. as well as physical activities that may erode a polymeric blendmatrix such as diffusion, abrasion, cracking, peeling, mechanicalbreakage, spinodal decomposition, etc. or any combination of thesechemical and physical activities.

The present invention provides a novel method for simultaneously andindependently determining both surface concentration of a drug anddegradation kinetics of degradable polymer/drug blend matrix by usingTime-of-Flight Secondary Ion Mass Spectrometry (ToF SIMS) measurements.The method can be used for in vitro studies of polymeric biomaterialssuch as those intended for use as drug delivery systems andsignificantly reduce the need of in vivo studies. However, whendesirable, the method can also be used for studies of in vivo polymerdegradation.

The invention provides a method for simultaneously determining surfaceconcentration of a drug in the blend matrix and reaction kinetics of adegradable polymer in a blend matrix comprising the following steps:

providing a polymer/drug matrix, initiating degradation of thepolymer/drug matrix, subjecting the degraded polymer/drug matrix to highmass and low mass ToF SIMS spectral analysis, identifying andquantifying oligomers from the high mass ToF SIMS over a period of time,identifying and quantifying surface drug from the ToF SIMS spectra overthe same period of time, and calculating the rate of formation of one ormore oligomers and the rate of change of surface concentration of drug.

In yet a further embodiment of the present invention, oligomers of ahydrolyzed biodegradable polymer are identified and quantified by thefollowing steps: providing a biodegradable polymer, initiatinghydrolytic degradation of the biodegradable polymer, subjecting thedegraded biodegradable polymer to high mass ToF SIMS spectral analysisand identifying and quantifying oligomers from the high mass ToF SIMSspectral analysis.

In certain embodiments of the invention herein the analysis is begun byobtaining specimens and mounting them on suitable sample holders. Anexample of a suitable specimen is a thin film specimen. Methods ofpreparing thin film specimens of the blend matrix include, for example,melt pressing, melt casting, spin coating, solvent casting, monolayerfilm production, lamination, etc.

Non-limiting examples of polymers useful in the practice of theinvention herein include polyesters, polyanhydrides, copolymers ofpolyesters and polyanhydrides and mixtures thereof. When the polymercomponent is a polyester, the polyester may be formed of poly(α-hydroxyacids), poly(β-hydroxy acids), poly(α-malic acids), pseudo poly(α-aminoacids), copolymers thereof and mixtures thereof. When the polymercomponent is a polyanhydride, the polyanhydride may be formed ofhomo-polyanhydrides of sebacic acid, homo-polyanhydrides of fumaricacid, random co-polyanhydrides of sebacic and fumaric acids, andmixtures thereof.

When the specimen is a blend matrix, the non-polymeric component can beany of one or more broad types of materials. Non-limiting examples ofthese non-polymeric materials would include: catalysts, chemicals, dyes,low molecular weight drugs, hydrophilic drugs, hydrophobic drugs,peptides, short chain length polypeptides and mixtures thereof.

As a reference ToF-SIMS spectrum for the untreated specimens, thesurface of the specimens is doped with, for example, Na⁺using 1 N NaClsolution, followed by suitable drying, for example, quick dry in vacuumat least for 24 hours. Other doping ions such as potassium and silvermay be suitable depending upon the specific application. The choice ofdopant is within the purview of one skilled in the art.

Degradation of the specimen can be performed by exposing the material tolytic solution,acids, bases, etc., abrading the specimen, solventimmersion, buffer solution immersion. Therefore, methods of degradingwould include solvating, desorbing, dissociating, hydrolyzing,dissolving, diffusing, abrading and combinations thereof The degradationcan take place at any suitable temperature, for example, roomtemperature, body temperature, etc.. When degradation is accomplished byhydrolyzing in buffer solution, the buffer solution can be varioussaline buffers having a pH between about 2.0 and about 12.0 andcontaining ions such as phosphate, acetate, carbonate, biphthalate, ormixtures thereof.

The specimen can be dried before and/or after degradation by processesincluding but not limited to: contacting the specimen with a gradedseries of dehydrating liquids; subjecting the specimen to microwaveenergy; subjecting the specimen to heat at ambient or sub-atmosphericpressures, e.g., drying oven at temperatures from about 35° C. to about85° C., or vacuum oven at temperatures from about 35° C. to about 85°C.; subjecting the specimen to sub-atmospheric pressure in the presenceor absence of a desiccant, e.g., closed container subjected to vacuumoptionally containing a desiccant such as anhydrous calcium chloride,anhydrous silica gel or the like; etc.

ToF-SIMS analysis can be performed with any appropriate instrument. Adescription of ToF-SIMS instruments can be found in “ToF-Sims, SurfaceAnalysis by Mass Spectrometry”, Eds. J. C. Vickerman and David Briggs;I. M. Publications, U. K. (2001).

To extract a MW average from the distribution of oligomeric degradationproducts, the spectra are analyzed using a conventional statisticalaveraging definition for the number average molecular weight (M_(n)).This is then converted to degree of polymerization (DP). For example, inthe case of PLLA, the average DP at time t is defined as the repeatingnumber of M_(n) of PLLA degradation products:

DP=(M _(n)−18)/(72)  (1)

where 18 is the mass of both end-groups and 72 is the mass of PLLArepeat unit. The kinetics expression for the surface degradation byhydrolytic chain scissions of PLLA linkages was derived as a pseudofirst-order reaction:

ln[(DP−1)/DP]=−k t +ln[(DP _(s)−1)/DP _(s)]  (2)

where DP and DP_(s) are the degrees of polymerization of hydrolysisproducts at time t and being first generated when degradation starts (>0hour), respectively. Therefore, the distribution of PLLA degradationproducts in high mass range over 600 m/z of ToF-SIMS can be converted tothe corresponding MWD function in two terms (N_(i) vs. M_(i)) of astatistical averaging MW calculation. Using the MWD function, theresults of both M_(n) and resultant semilog terms, ln[(DP−1)/DP], forkinetics can be calculated. The semilog plots are shown in FIG. 2 as afunction of hydrolysis time at two pHs. In these examples, the slopeobtained from the linear fitting represents the rate constant ofhydrolytic PLLA degradation at the surface of Ph₃N/PLLA (20:80 wt%)blend matrices. The results of linear regression of the degradationkinetics represent the characteristic induction phase of thebiodegradable poly(α-hydroxy acid) bulk erosion profile.

Data is presented for the hydrolytic degradation of six biodegradablepolymers, involving two important classes of biodegradable polymers, inparticular polyesters and polyanhydrides, and both homopolymers andrandom copolymers, using ToF SIMS. Upon hydrolytic degradation of thesepolymers, low molecular weight oligomers generated during the hydrolyticdegradation can be directly detected in ToF SIMS spectra. Further datais presented for the degradation of a model drug delivery system.

The following examples are presented for illustrative purposes and arenot to be construed as restricting the claim in any way whatsoever.

Example 1

Poly(glycolic acid) (PGA) was obtained from the Davis & Geck Division ofAmerican Cyanamid Company; Poly(l-lactic acid) (PLA) (weight averagemolecular weight 93,000) and poly(dl-lactic-co-glycolic acid) (PLGA)(50:50, molecular weight 50,000-75,000) were purchased from SigmaChemical Company (St. Louis, Mo.); Poly(fumaric-co-sebacic acid) (PFS)(50:50, average molecular weight 3,046, 20:80, average molecular weight6,500) and poly(sebacic acid) (PSA, average molecular weight 12,000)were supplied by Brown University; and Physiological solution ISOTON® IIwas purchased from Coulter Diagnostics in Hialeah, Fla.

PLLA (mol. wt. 100,000) was purchased from Polysciences, Inc.(Warrington, Pa.) and Ph₃N (98%) was purchased from Aldrich (St. Louis,Mo.). A physiological electrolyte buffer solution, ISOTON® II (pH 7.4 at25° C.), was purchased from Coulter Diagnostics (a division of CoulterElectronics, Inc. Hialeah, Fla.). Sodium carbonate buffer solution (pH10.0 at 25° C.) was prepared with buffer concentrates (DILUT-IT®)purchased from J. T. Baker Inc. (Phillipsburg, N.J.). HPLC gradechloroform (CHCl₃) from Aldrich was used for the preparation of ˜2%(w/v) Ph₃N/PLLA mixture solutions.

PGA, PLA and PLGA samples were prepared by melt-pressing on aluminumfoil. Prior to each melt-press, thick PGA plates were cut to smallpieces and washed with an ultrasonic cleaner (from Branson CleaningEquipment Company, model 5-52) in hexane and chloroform for 10 minuteseach. Aluminum foil was pre-cleaned with chloroform. PLA and PLGA wereused as received. Samples were pressed at about 200° C. to about 1 mm ofthickness. The aluminum foil was peeled off and hydrolysis treatment wasconducted immediately.

Polyanhydride samples were prepared by melting polymers on aluminum foilat their melting temperatures. Polymer samples, about 1 mm thick, werepre-cleaned in hexane and vacuum-dried prior to melt. The aluminum foilwas peeled off for PSA samples.

Ph₃N/PLLA blend matrices from the ˜2% (w/v) mixture CHCl₃ solutions werespin-coated onto 10×10 mm glass plates at 2000 rpm for 60 seconds usinga Headway Research Inc. Model EC 101 spin-coater. The thickness wasmeasured to ca. 390 nm using a profilometer (Alpha-step® 500, TencorInstruments). The morphology was clear and flat at 700X magnification ofscanning electron microscopy (SEM) photomicrographs. In order to confirmthe homogeneous distribution of Ph₃N in the matrices, theback-scattering image was measured using a Hitachi S-4000 scanningelectron microscope equipped with a back-scattered electron detector.The image of back-scattered electrons was homogeneous, which supportsthe interpretation that the drug/polymer blend matrices werehomogeneous; i.e., no microphase domains of drug were detected in thepolymer.

The amount of Ph₃N in Ph₃N/PLLA (20:80 wt%) blend matrices wascalculated to ca. 0.73 μmole/sample from dissolving the entire film byimmersing a sample specimen (film+substrate) in 24 ml of CHCl₃ solutionat least for 24 hours. The in vitro hydrolysis of Ph₃N/PLLA (20:80 wt%)blends was conducted at 37.0±0.2° C. in two pH saline buffer solutions(pH 7.4 and pH 10.0) in order to regulate the local autocatalytic effectof carboxylic acid end groups generated during the treatment. Each blendmatrix was immersed in a separate vial pre-filled with 24 ml of bufferedsolution and reaction vials were placed in an isothermal water bath(Fisher Circulator Model 73) for the predetermined time. All matricesafter the allotted times were vacuum-dried at ambient temperature atleast for 24 hours before being analyzed. The pH values and the extentof Ph₃N diffusion into the buffers were examined after the hydrolysistreatment using a pH meter (Digital Ionalyzer Model 501 of OrionResearch Inc.) and a Milton Roy Spectronic 1201 UV spectrophotometer,respectively. Little change in pH value (±0.1 pH units) was observed andno detectable UV absorption of Ph₃N was measured from the analysis ofbuffers, from which it can be postulated that the diffusion effect of adrug is minimized in the present model system of drug delivery and theaccumulation rate of Ph₃N at the surface of blend matrices representsthe amount of a drug available for release of the hydrophobic drug as afunction of hydrolysis time.

The hydrolytic degradation of all non-blend matrix polymers was carriedout in a physiological buffer solution, ISOTON® II (pH=7.4), at 37.0° C.Each sample was immersed in a separate vial prefilled with 14 ml ofISOTON® II solution and sealed airtight. The reaction vials wereimmersed in a temperature bath for pre-determined periods depending onthe sample's sensitivity to hydrolytic degradation and other propertiessuch as molecular weight and hydrophobicity of the polymer. Thehydrolysis time for each polymer was chosen so that the molecular ionsof hydrolysis products were observed with good peak intensity. Thehydrolysis times of all non-blend polymer samples used in this study arelisted in Table 1.

TABLE 1 Initial Molecular Weight and Time of Hydrolysis ReactionsHydrolysis Time Polymer Molecular Weight (in hours) PGA NA 1 PLA 93k 30PLGA 50-75k 24 PSA 12k 24 PFS 20:80 6.5k  3 PFS 50:50  3k 2

Samples after the hydrolysis treatment were vacuum-dried at ambienttemperature, and stored in sealed vials filled with argon until ToF SIMSanalysis was performed.

ToF SIMS analysis was conducted on a Physical 20 Electronics 7200time-of-flight secondary ion mass spectrometer equipped with a pulsedcesium ion gun and a channel plate detector. The primary ion gun wasoperated at 8 keV in all spectral acquisitions. The static mode was usedin all acquisitions with the primary ion current of 0.3 pA. The pulsewidth of primary ion current was 1.0 ns. The total ion dosage in eachspectral acquisition did not exceed 1×10¹¹ ion/cm². An electronneutralizer was operated during all spectral acquisitions in pulse modeat low electron energy with a target current under 1 μA for chargecompensation. A time resolution of 1.25 ns per step was used for goodsignal-to-noise ratio at high m/z range. The pressure of the mainchamber was kept between 10⁻⁸ and 10⁻¹⁰ torr for each analysis. Datareduction was performed using Physical Electronics TOFPak™ software.

Example 2 ToF SIMS Results of Hydrolyzed Polyesters

Ions from polymer chain fragments, with exponentially deceasingintensity, are normally observed in the high mass range ToF SIMS spectraof thick film polymer samples, unless the sample film is prepared asmonolayers deposited on metal substrates. Oligomeric ion distributionsare not normally observed for thick films due to the entanglement of thelong chain molecules in polymer samples. Random chain scission occurswhen samples are bombarded by primary ions, which transfers energy topolymer chains near the surface. This process generates fragment ions inToF SIMS spectra with the characteristic of exponentially decreasedintensity with few meaningful peaks due to the decreased possibility ofproducing high mass fragments.

Upon hydrolysis, polymer chain-lengths are reduced gradually until theoligomers become small enough to desorb from the polymer surface anddissolve in the surrounding liquid phase, where they continue tohydrolyze; yielding monomers as the ultimate reaction products. On thesurface of a degraded polymer, the entanglement of thedegradation-generated oligomers is greatly reduced because of thedecrease in molecular chain-length. During ToF SIMS measurements, lowmolecular weight oligomers become easier to desorb from the samplesurface upon the bombardment of primary ions. Therefore, whendegradation occurs at polymer sample surfaces, intact molecular ions ofthe degradation products can be observed in relatively high mass rangeof ToF SIMS spectra.

ToF SIMS Results of Hydrolyzed PGA

FIG. 3 shows the high mass portion (600 D to 2000 D) of ToF SIMS spectraof PGA hydrolyzed for one hour. Before the hydrolysis treatment,essentially nothing can be observed in this range except a noisybackground. Upon hydrolysis, a peak pattern characterized by thedifferences due to the mass of the repeat unit of PGA was observed.

All the ions of the major peaks in FIG. 3 have the structure of[nG+H₂O+Na]⁺, where G stands for the repeat unit of PGA. This indicatesthat the ions detected from the hydrolyzed PGA sample are in the intactmolecules of the hydrolytic degradation product. The sodium ion comesfrom the external buffer treatment solution and participates in theprocess of secondary ion formation as an ionization assisting agent. Itwill be seen that in all samples studied, sodium plays an important rolein the ionization process and all species detected in this seriescontain at least one sodium ion each. It was also observed that, whenthe concentration of potassium is high enough to promote secondary ionformation of hydrolysis products, a set of molecular ion peaksassociated with potassium could be present simultaneously with theseries of peaks associated with the sodium ion.

Molecular ions of up to 33 repeat units were observed in ToF SIMSspectra of some samples of hydrolyzed PGA. Small oligomers detected inthis study can be traced down to the final hydrolysis product, forexample the single glycolic acid molecule associated with one sodiumion. Table 2 lists the full range of molecular ions that have beendetected in this study, with the ions shown in FIGS. 3 and 4 listed inbold.

TABLE 2 Hydrolysis Products of BOA and PLA Observed in ToF SIMS SpectraNumber of monomers PGA m/z PLA composition nG + H₂O nL + H₂O 1 99 113 2157 185 3 215 257 4 273 329 5 331 401 6 389 473 7 447 545 8 505 617 9563 689 10 621 761 11 679 833 12 737 905 13 795 977 14 853 1049 15 9111121 16 969 1193 17 1027 1265 18 1085 1337 19 1143 1409 20 1201 1481 211259 1553 22 1317 1625 23 1375 1697 24 1433 1769 25 1491 1841composition nG + H₂O nL + H₂O 26 1549 27 1607 28 1665 29 1723 30 1781 311839 32 1879 33 1955 34 2013 35 2071 36 2129 37 2187 38 2245 39 2303 402361 41 2419 42 2477 43 2535 44 2593 45 2651

In addition to the wide distribution of molecular ion peaks, a crest ofthe molecular ion peaks can also be seen. This crest (1) was firstobserved in the one hour hydrolysis sample spectra (FIG. 3) at 1400 D to1500 D, and (2) became more pronounced when it moved to the low massrange gradually as the hydrolysis time increased. This crest representsthe most probable molecular weights of hydrolysis products at theparticular reaction time. The hydrolytic degradation kinetics can beexplored using the data from the ToF SIMS analysis.

ToF SIMS of Hydrolyzed PLA

PLA has the same main chain backbone as PGA plus a methyl group as aside chain. The presence of the methyl group, however, significantlychanges the properties of the ester carbon as well as the bulk polymerproperties such as morphology and hydrophobicity. These changes arereflected in the characteristic rates of hydrolytic degradation andconsequently in the ToF SIMS spectra of hydrolyzed samples. FIG. 3 showsthe high mass portion (from 500 D to 2000 D) of ToF SIMS spectra of PLAdisc samples hydrolyzed for 30 hours. The star marked peaks are the mostintense peak in each repeat pattern. As in the spectra of PGA, eachrepeat pattern corresponds to one repeat unit of PLA. The intervalsbetween each of the star marked peaks are 72.02 m/z, exactly the mass ofone PLA repeat unit.

Molecular ion peaks are observed from the surface of hydrolyzed PLA fromthe final hydrolysis product (for example, the single lactic acidmolecule) up to the oligomer with 25 repeat units of PLA. The intensityof the low mass species (not shown in the figures) becomes lower andlower quickly, this is likely due to the increased diffusibility of thelow mass hydrolysis products from the solid sample surface to thehydrolysis solution and the increased solubility as the molecular weightis reduced.

A remarkable difference between PLA and PGA is the hydrolyticdegradation rate. The spectra shown in FIG. 4 are PLA hydrolyzed for 30hours under the same hydrolysis conditions as that for PGA. For PLAsamples hydrolyzed in shorter times, good peak patterns of hydrolysisproducts could not be observed. In addition, there is no peak crestobserved at this hydrolysis time, as seen in the ToF SIMS spectra ofhydrolyzed PGA (FIG. 3) The intensity of molecular ion peaks isexponentially decreasing as the m/z increases.

ToF SIMS of Hydrolyzed PLGA

FIG. 5 shows the high mass portion (400 D to 2000 D) of the ToF SIMSspectrum of PLGA 50—50 random copolymer hydrolyzed for 24 hours. Thepattern of the spectra is obviously more complicated than both the PGAand PLA spectra. Each of the peaks in FIG. 5 consists of a group ofpeaks (see the inset in FIG. 5). The intervals between the groups areessentially 14 D indicating the repeat pattern is governed by thestructural difference between monomeric lactic and glycolic acids. Inaddition, the most intense peak in each group shifts gradually towardslower m/z so that the overall interval of the repeat pattern is not theexact m/z of one CH₂ group over the full range shown in FIG. 5. Thiscomplicated pattern can be understood by considering all possiblecompositions of hydrolysis products of this random copolymer. Table 3tabulates the m/z values of all possible molecular ion compositions interms of m/z for low molecular weight oligomers of the hydrolysisproducts up to 15 PLA repeat units and 12 PGA repeat units.

TABLE 3 Theoretically Predicted PLGA Oligomers of Hydrolysis Products*

*The hydrolysis products are tabulated in m/z with the ion compositionof [xL + yG + H₂O + Na] +, where L and G represent the monomeric repeatunit of lactic acid or glycolic acid, respectively. Ions in borderedcells are shown in FIG. 6. Ions in bold become too low in intensity.**The number of lactic acid monomers in each oligomer molecule is shownin rows and the number of glycolic acid monomers is shown in columns.

These ions represent the structure of intact molecules which would thenbe cationized with at least one sodium ion each. All molecular ionslisted in Table 3 are observed in the ToF SIMS spectra except those withhigh composition ratios of PGA over PLA repeat units. This is becausePGA is more sensitive to hydrolysis than PLA. To illustrate this fact,ions in the framed cells (oligomer molecular ions from 800 D to 1000 D)are shown and marked with m/z values in FIG. 6. It shows that most peaksshown in the spectra are molecular ion peaks with the most intense peakshifts to the left in each cluster due to the composition of themolecular ions. In fact, the most intense peak in each cluster is alwayscorresponding to the ion with the smallest number of glycolic acidrepeat units and the largest number of lactic acid repeat units. Thepeak intensities for the ions in the shaded cells of Table 3 consistingof larger ratio of glycolic acid repeat units than the ions to theirleft, become too low to be detected.

The relative intensity of molecular ion peaks also serves as anindication of the relative hydrolysis rate of the two components of thecopolymer. PGA hydrolyzes faster than PLA as observed in thehomopolymers of PGA and PLA; therefore, fewer PGA repeat units remain inthe hydrolysis products. No oligomers with only PGA repeat units wereobserved while oligomers with pure PLA were observed as the most intensepeak in its group (peaks of 833, 905 and 977 D in FIG. 6, for example).

FIG. 7 exemplifies the composition of the molecular ion peaks bycomparing one of the peak groups with the theoretically calculatedspectra. FIG. 7a is a small portion of the spectrum shown in FIG. 6 andFIG. 7b is the corresponding molecular ion peak region theoreticallycalculated using Googly (Copyright 1994, Andrew Proctor), taking theelemental isotopic abundance into account. The match in peak positionand relative intensity between the experimentally recorded spectra andthe theoretically predicted one supports the assignment of the peaks.FIG. 6 indicates that all major peaks in the ToF SIMS spectra ofhydrolyzed PLGA copolymer are intact molecular ions of the hydrolysisproducts.

PGA is the simplest biodegradable poly(α-hydroxy acid) with highcrystallinity and hydrophilicity. PLA is only one methyl group differentfrom the structure of PGA as the side-chain on the α-carbon, whichcauses a remarkable change in its properties from PGA, in addition tothe formation of two monomeric enantiomeric structures, and copolymersof different tacticities. For example, the crystallinity of both P(d)LAand P(l)LA is lower than PGA and the presence of the methyl group in PLAsignificantly decreases its reactivity toward ester hydrolysis mechanismdue to the electron donating effect, resulting in the global decrease inhydrophilicity. As a result, the increased hydrophobicity can reasonablyexplain the relatively slower hydrolytic degradation of PLA and makesPLA dissolve well in common organic solvents in contrast to PGA, whichis soluble only in hexafluoroisopropanol. Hence, these properties caninfluence not only each own hydrolytic degradation properties, but alsothe fragmentation process upon the bombardment of the primary ions.

Therefore, the difference in each hydrolytic degradation rates betweenPLA and PGA can be supported by the observation from ToF SIMS spectra ofhydrolyzed PLGA 50:50 copolymer. As shown in FIG. 6, peaks consisting ofmore PLA repeat units and less PGA repeat units are always even moreintense than those consisting of more PGA repeat units; indicating thatPLA segments are less reactive to hydrolysis than PGA segments. Theoverall trend of molecular ion peak intensities of the hydrolyzed PLGAcopolymer is similar to that of hydrolyzed PLA spectra (FIG. 4).

Example 3 ToF SIMS of Hydrolyzed Polyanhydrides

Polyanhydrides are significantly different from polyesters in that theanhydride linkage in the backbone is more vulnerable to attack by waterthan the ester bond. This leads to faster hydrolysis rates forpolyanhydrides and causes a narrow molecular weight distribution of thehydrolysis products. FIG. 8 shows the ToF SIMS spectra of hydrolyzed PSAfrom 400 D to 1200 D. Molecular ion peaks are the most dominant one ineach repeat pattern, indicating that the anhydride bond is far easier tobreak than the alkyl chain. Intact molecular ions observed are from thesingle sebacic acid molecule up to the oligomer of six PSA repeat units.The single sebacic acid molecule, which is the first member in theseries of hydrolysis products, however, is small enough to be dissolvedin the hydrolysis solution. Hence, very few of them stay on the surfaceof samples after the hydrolysis experiments. Therefore, the firstsignificant molecular ion peak is the one that consists of two PSArepeat units. All observed oligomers of the hydrolysis products arelisted in Table 4 in the form of actually observed ions, consisting ofthe intact molecules attached with a sodium ion.

TABLE 4 Molecular Ion Peaks Observed in Hydrolysis Products ofPolyanhydrides # of repeat m/z 1 225 225 225 2 409 409 409 3 593 593 5934 777 777 777 5 961 961 6 1145  *Molecular ion peaks of the finalproducts are very low intensity, not shown in the figures.

Remarkably different from the polyesters, the intensity of molecular ionpeaks drops quickly and exponentially. It can be seen from the spectrain FIG. 7 that there would be no species larger than the six-repeat-unitoligomer detectable on the sample surface.

ToF SIMS of Hydrolyzed PFS Copolymers

Two random copolymers of PFS have been studied, in particular 50:50 and20:80 by weight percentage of fumaric acid to sebacic acid ratio. Theinitial molecular weight of 50:50 PFS sample is about 3000 by numberaverage molecular weight, and that of 20:80 PFS sample is about 6000.Considering the molecular weights of the repeat units being 98 and 184for furmaric acid and sebacic acid, respectively, the degree ofpolymerization is quite low for both copolymers, approximately 20 forthe 50:50 copolymer and 36 for the 20:80 copolymer. It was expected thatunreacted oligomers might be detected in the medium mass range of ToFSIMS spectra of unhydrolyzed samples. FIG. 9 shows the ToF SIMS spectraof 20:80 copolymer before hydrolysis from 350 D to 1050 D. As it isexpected, significant ion series were detected up to 1000 D. The ionsequence of 467 D, 651 D, 835 D and 1019 D (framed-number marked in FIG.9) has the composition of [F+nS+H]⁺, in which F and S represent therepeat unit of fumaric acid and sebacic acid, respectively. However, inaddition to the fragment ion peaks, the molecular ion peak series of 409D, 593 D, 777 D and 961 D are also present. The composition of thisseries conforms to the ion structure [nS+H₂O+Na]⁺, indicating that thecopolymer has already partially hydrolyzed during storage. Note that therelative intensity of the two series changes as the m/z increases. Thefact that the relative intensity of the molecular ion peak series to thefragment series increases as the m/z increase indicates that thedistribution of molecular oligomer ions is independent from the fragmention distribution.

Upon hydrolysis treatment, the fumaric acid repeat unit could not bedetected from any hydrolysis products. FIG. 10 shows the ToF SIMSspectra of 20:80 copolymer from 400 D to 1200 D. The spectra ofhydrolyzed PFS copolymer are almost the same as that of hydrolyzed PSA,indicating that fumaric acid component in the random copolymer chainsequence is far more sensitive to a hydrolysis environment, andhydrolyzes faster than the PSA sequences. The marked peaks in FIG. 10have exactly the same ion composition as the hydrolysis products of PSA.The difference between the spectrum and that of the product ofhydrolyzed PSA is that the largest molecule detected in 20:80 PFScopolymer has five sebacic acid repeat units while the largest moleculedetected in PSA has six sebacic acid repeat units, in spite of theshorter hydrolysis time for the copolymer samples. This is an additionalindication of the faster hydrolysis property of the PFS copolymercompared to the homopolymer of PSA.

Similar results were observed for the 50:50 PFS copolymer. FIG. 11 showsthe ToF SIMS spectra of the hydrolyzed sample from 400 D to 1200 D.Similar to the 20:80 sample, only low molecular weight oligomers of PSAwere observed and products are more narrowly distributed to lowermolecular weights. The largest oligomer molecule observed in the twohour hydrolyzed sample of this copolymer has only four sebacic acidrepeat units. In addition, the intensity of the molecular ion peaksdecreases much faster than the 20:80 copolymer and the homopolymer ofPSA, indicating higher hydrolysis rate is associated with higher fumaricacid content.

Although there was no fumaric component detected in the hydrolyzedsample of both the two PFS copolymers, fragment ions from fumaric acidrepeat units were indeed detected in all cases, indicating that thefumaric acid repeat unit is released from the polymer chain sequence asa whole unit during the hydrolytic degradation. FIG. 12 shows thefragment peaks of fumaric acid single unit before and after hydrolysisfor two hours, Peaks of 97 D, 98 D and 99 D are the ions of [F−H]⁺,F⁺and [F+H]⁺, respectively. The increased relative intensities of F+toother peaks may also indicate the contribution of hydrolytic cleavage offumaric acid bonds.

Both PSA and PFA (poly(fumaric acid)) are highly crystalline materials.It has been determined that the crystallinity of homopolymers of PSA andPFA are 66%, respectively. The crystallinity of their copolymersdecreases depending on the composition of the copolymer, but it is nolower than 38% for all compositions. The samples of the polyanhydridesstudied in this work, therefore, cannot be made by solution-casting. Oneof the concerns for the melt-cast sample preparation procedure is thepossibility of oxidation or cross-linking of the double bond in fumaricacid at elevated temperatures. There is no evidence found, however, thatthe double bond in fumaric acid has been severely changed. The intensefragments of fumaric acid at 97 D, 98 D and 99 D, corresponding to[F−H]⁺, F⁺and [F+H]⁺, are evidence of the existence of an abundance ofunreacted fumaric acid structures (FIG. 12a). This structure was alsodetected in high intensity after the hydrolysis treatments (FIG. 12b),suggesting the basic repeat units of fumaric acid was not changed duringthe hydrolysis reaction either.

However, fragment ions containing multiple fumaric acid repeat unitswere never detected in this study. In the ToF SIMS spectra of PFSsamples without hydrolysis treatments, only one fumaric acid repeat unitwas detected in fragment sequences containing fumaric acid, whereasfragments with up to five sebacic acid repeat units were detected. Themolecular ion peak series of hydrolysis products were found in bothspectra of 20:80 and 50:50 copolymer samples before hydrolysistreatments. These molecular ion peak sequences consist of only sebacicacid monomers, suggesting that the anhydride bond of fumaric acid ismore sensitive to hydrolysis than the anhydride bond of sebacic acid.There are two factors each may play an important role in this issue. Oneis the hydrophilicity. Sebacic acid contains a highly hydrophobicaliphatic structure while the structure of fumaric acid is highlyhydrophilic. This may result in the hydrolytic degradation duringstorage to occur selectively at the fumaric acid sequence. The otherfactor is the conjugative property of the fumaric acid structure. Withtwo carboxylic acid groups bridged by a double bond, fumaric acid formsa conjugated structure. This conjugated system increases the reactivityof the carboxylic acid carbon towards nucleophilic reactions.

The differences in hydrolytic degradation rates among the threepolyanhydrides can be seen by the hydrolysis time and the hydrolysisproducts illustrated in the ToF SIMS spectra. Table 4 lists allmolecular ions detected in the hydrolyzed samples of all threepolyanhydrides. The largest molecule of PSA hydrolysis products has sixsebacic acid repeat units while the 20:80 copolymer has five and the50:50 copolymer has four, although shorter hydrolysis time were used forthe PFS copolymers. Furthermore, the intensities of the most intensemolecular ion peaks are about 3500, 2000 and 800 for 50:50, 20:80 andthe homopolymer of PSA, respectively. Therefore, the molecular ion peaksdecrease faster for copolymers that have higher fumaric acid content.

Example 4 Information for Kinetics Analysis

As mentioned above in Example 2, a crest of the molecular ion peaksexists which grows and moves toward the low mass end when the hydrolysistime increases. Under the assumption that this crest represents the mostprobable molecular weight distribution of the hydrolysis products, theaverage molecular weight of the hydrolysis products can be calculatedfrom the ToF SIMS spectra. The average molecular weight obtained fromToF SIMS is a function of hydrolysis time. A linear relationship betweenthe apparent molecular weight and the hydrolysis time has been observed.This observation indicates that the ToF SIMS spectra of hydrolyzedsamples carry information about the hydrolytic degradation process ofthe polymer, which can be used in kinetics and mechanism analysis ofhydrolytic degradation of the polymer. Based on this, ToF SIMS studiesof the degradation kinetics can be carried out.

The size of the largest molecule of the hydrolysis products ofpolyanhydrides decreases as the content of fumaric acid in the copolymerincreases, and the molecular ion peak intensity decreases fasteraccordingly. This is directly related to the hydrolytic degradationproperty of the polymer. Also, the intensity of the molecular ion peaksdecreases exponentially. When the logarithm of the intensity of themolecular ion peak is plotted versus the degree of polymerization, agood linear relationship exists, as shown in FIG. 13. The slope of theplot reflects how fast the molecular ion peak intensity decreases, andwhere the line crosses with the line of Log(y)=0, which indicates themolecule of the size marked by the cross point could not practicallyexist anymore. The slopes of the straight lines have a proportionalcorrelation with the hydrolytic degradation rates. Therefore, thehydrolytic degradation rate can be quantitatively described by thisparameter.

Example 5

To determine the concentration of Ph₃N at the surface, peak intensitiesrepresentative of the drug and polymer components from the spectrum ofuntreated Ph₃N/PLLA blend matrices were integrated over two mass ranges:53˜59 D for [C₃H₄O]^(·+)=56 D of PLLA and 241˜267 D for [MH]⁺=246 D ofPh₃N (M=Ph₃N in this example). In this study, the fragment ion peak,[C₃H₄O]^(·+)=56.0264 D, from the PLLA repeat unit was used as aninternal standard to quantify the surface concentration of Ph₃N. Theratio of [MH]⁺peak intensity divided by the peak intensity of[C₃H₄O]^(·+) was related to the total amount of Ph₃N incorporated in thematrices, and was considered as proportional to the surfaceconcentration of Ph₃N. A standard calibration curve was developed (notshown). Good linearity (R²=0.9984, A=8.92E-4 in Y-0.00768=AX) wasobtained from a concentration ratio (X) of 10:90 drug to polymer wt% upto 40:60.

A peak at 246 m/z in the spectrum of pure PLLA was observed to overlapwith [MH]⁺=246 D. The relative intensities, [a peak at 246 m/z]/[C₃H₄O]^(·+), of pure PLLA matrices were measured after the hydrolysisunder two different pH buffered conditions for 24 hours, respectively:2.62E-3 for pH 7.4 and 2.59E-3 for pH 10.0. They indicate that therelative intensity of the peak at 246 m/z ratioed to the intensity of[C₃H₄O]^(·+) in pure PLLA is independent of pH and hydrolysis time.Therefore, the change in the ratio of intensities, 246 m/z divided by 56m/z, was used as a measure of release profiles that represent a changein surface concentration of Ph₃N. The surface concentration of Ph₃N fromthe 20:80 wt% blend matrices has been measured as a function ofhydrolysis time at two buffered pHs and compared to the correspondingmeasured relative intensity ratio from a series of Ph₃N/PLLA blendmatrices in FIG. 14 for evaluating the cumulative amount of Ph₃N. Thecurves were fit with an empirical exponential expression,([Ph₃NH]^(+/[C) ₃H₄O]^(·+))=9.37E-3 +Ae{circumflex over ( )}(t/7.94):A_(pH10)=−9.36E-5 for pH 10.0 and A_(pH7.4) =−1.58E-5 for pH 7.4. (RightY axis) Surface concentration of Ph₃N was obtained from the standardcalibration curve for surface concentration of Ph₃N. The extent ofchange in accumulation rate of Ph₃N (A_(pH10)=5.92×A_(pH7.4)) is morethan two times greater than the corresponding change in hydrolyticdegradation rate of PLLA (k_(pH10)=2.67×k_(pH7.4)) at the surface ofPh₃N/PLLA (20:80 wt%) blend matrices.

These data demonstrate that the oligomers desorb from the sample surfaceupon the bombardment and ionization, in the form of intact molecules,usually attached with an alkali metal ion. In most cases, the molecularion peak is the most intense peak in each repeat pattern of ToF SIMSspectra of hydrolyzed polymers.

From the foregoing, it will be obvious to those skilled in the art thevarious modifications in the above-described methods, and compositionscan be made without departing from the spirit and scope of theinvention. Accordingly, the invention may be embodied in other specificforms without departing from the spirit or essential characteristicsthereof. Present examples and embodiments, therefore, are to beconsidered in all respects as illustrative and not restrictive, and allchanges which come within the meaning and range of equivalency of thespecifications are therefore intended to be embraced therein.

We claim:
 1. A method for simultaneous determination of surface drugconcentration and reaction kinetics of degradation of a drug-polymermatrix comprising a drug and a polymer comprising the steps of:providing the drug-polymer matrix; initiating degradation of thedrug-polymer matrix; subjecting the degraded drug-polymer matrix to ToFSIMS spectral analysis; identifying and quantifying oligomers on thesurface of the drug-polymer matrix from the ToF SIMS spectra as afunction of time; identifying and quantifying surface drug concentrationfrom the ToF SIMS spectra as a function of time; and, calculating therate of formation of one or more oligomers on the surface of thedrug-polymer matrix, wherein the rate of formation of one or moreoligomers is indicative of the rate of degradation of the polymer. 2.The method of claim 1 wherein the polymer in the drug-polymer matrix isselected from the group consisting of polyesters, polyanhydrides,copolymers of polyesters and polyanhydrides and mixtures thereof.
 3. Themethod of claim 2 wherein the polyester in the drug-polymer matrix isselected from the group consisting of poly(α-hydroxy acids),poly(β-hydroxy acids), poly(α-malic acids), pseudo poly(α-amino acids),copolymers thereof and mixtures thereof.
 4. The method of claim 2wherein the polyanhydride in the drug-polymer matrix is selected fromthe group consisting of homo-polyanhydrides of sebacic acid,homo-polyanhydrides of fumaric acid, random co-polyanhydrides of sebacicand fumaric acids, and mixtures thereof.
 5. The method of claim 1wherein the step of initiating degradation comprises solvating thedrug-polymer matrix.
 6. The method of claim 1 wherein the step ofinitiating degradation comprises desorbing the drug-polymer matrix. 7.The method of claim 1 wherein the step of initiating degradationcomprises dissociating the drug-polymer matrix.
 8. The method of claim 1wherein the step of initiating degradation comprises hydrolyzing thedrug-polymer matrix.
 9. The method of claim 8 wherein the step ofhydrolyzing comprises contacting the drug-polymer matrix with at leastone saline buffer having a pH between about 2.0 and about 12.0, whereinthe saline buffer contains an ion selected from the group consisting ofphosphate, acetate, carbonate, biphthalate and mixtures thereof.
 10. Themethod of claim 1 wherein the step of initiating degradation comprisesdissolving the drug-polymer matrix .
 11. The method of claim 1 whereinthe step of initiating degradation comprises oxidizing the drug-polymermatrix.
 12. The method of claim 1 wherein the step of initiatingdegradation comprises reducing the drug-polymer matrix.
 13. The methodof claim 1 wherein the step of initiating degradation comprisesphotolysing the drug-polymer matrix.
 14. The method of claim 1 whereinthe step of initiating degradation comprises diffusing the drug-polymermatrix .
 15. The method of claim 1 wherein the step of initiatingdegradation comprises abrading the drug-polymer matrix.
 16. The methodof claim 1 wherein the step of initiating degradation comprises crackingthe drug-polymer matrix.
 17. The method of claim 1 wherein the step ofinitiating degradation comprises peeling the drug-polymer matrix. 18.The method of claim 1 wherein the step of initiating degradationcomprises mechanically breaking the drug-polymer matrix.
 19. The methodof claim 1 wherein the step of initiating degradation comprisesspinodally decomposing the drug-polymer matrix.
 20. The method of claim1 further comprising: preparing a standard calibration curve;determining a ratio of a non-polymeric component peak intensity to apolymeric component peak intensity; and comparing the ratio to thestandard calibration curve.